A Novel high resolution MS platform for high-throughput screening of G protein-coupled receptors

用于高通量筛选 G 蛋白偶联受体的新型高分辨率 MS 平台

• 批准号: 10636377
• 负责人: Jon Jacobs
• 金额: $ 34.23万
• 依托单位: BATTELLE PACIFIC NORTHWEST LABORATORIES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-15 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10636377
• 关键词: Adaptor Signaling Protein; Address; Agonist; Amino Acid Motifs; Amino Acids; Antibodies; Biological; Biological Assay; Biology; CXC chemokine receptor 3; CXCR3 gene; Cell physiology; Clinical; Collection; Complex; Coupled; Data; Data Analyses; Detection; Development; Dimensions; Disease; Dose; Drug Screening; Drug Targeting; FDA approved; Family; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Proteins; Heterotrimeric GTP-Binding Proteins; Inflammatory; Ions; Isomerism; Ligands; Mass Spectrum Analysis; Membrane; Modernization; Pathway interactions; Pattern; Peptide Library; Peptides; Pharmaceutical Preparations; Pharmacology; Phosphopeptides; Phosphorylated Peptide; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Process; Protein Region; Proteins; Proteomics; Reagent; Reproducibility; Research; Resolution; Role; Sampling; Signal Transduction; Site; Specificity; Spectrometry; Speed; Structure; System; T cell regulation; T-Cell Activation; T-Lymphocyte; Technology; Testing; Time; Transducers; Travel; United States National Institutes of Health; antagonist; beta-arrestin; cell preparation; chemokine; computerized data processing; design; drug development; drug discovery; experience; high throughput screening; improved; informatics tool; ion mobility; mass spectrometer; novel; novel strategies; overexpression; phosphoproteomics; programs; receptor; recruit; response; screening; small molecule; small molecule libraries; ultra high resolution

Summary: Mass spectrometry (MS) has proven invaluable in studying the mechanisms of cellular signaling as MS platforms can directly provide amino acid residue site-specific phosphorylation data compared to traditional antibody-based approaches. However, limitations exist in current MS approaches in generating confident site-specific phosphorylation quantification. This is particularly evident in complex multi-phosphorylated protein motifs, where the detection of isomeric multi-phosphorylated peptides easily overwhelms any prediction scoring approach that is simply based upon the fragmentation spectra. There are many biological examples of hyperphosphorylated regions, where they are associated with receptor/ligand interactions, including G-protein coupled receptors (GPCRs), membrane receptors that are the most common targets for FDA-approved drugs. For accurate site-specific quantification of protein hyperphosphorylation we propose a transdisciplinary approach using ultrahigh resolution Ion Mobility Separation (IMS) integrated with highly accurate and sensitive MS and MS/MS spectra to enable the confident characterization of hyperphosphorylated GPCR ensembles with greatly improved sensitivity, and speed. We will use multi-level Structures for Lossless Ion Manipulations (SLIM) technology (SLIM-Orbitrap platform) to fully characterize phosphorylation of GPCR/antagonist interactions utilizing CXCR3, which plays a central role in inflammatory diseases through its regulation of T cell function as an initial test case. We plan to first integrate ultrahigh resolution IMS with an advanced Orbitrap MS platform for unambiguous decoding of hyperphosphorylated sites, evaluate the SLIM-Orbitrap MS platform for resolving hyperphosphorylated protein regions, and finally, perform comprehensive site-specific phosphoproteomics for GPCRs through screening of activated T cells with dose-responses of chemokine and small-molecule CXCR3 biased agonists.

摘要： 质谱 (MS) 已被证明在研究细胞机制方面具有无价的价值。 MS 平台的信号传导可以直接提供氨基酸残基位点特异性磷酸化 与传统的基于抗体的方法相比的数据。然而，目前的技术还存在局限性 MS 方法可以产生可靠的位点特异性磷酸化定量。这是 在复杂的多磷酸化蛋白质基序中尤其明显，其中检测 异构多磷酸化肽很容易压倒任何预测评分方法 简单地基于碎片光谱。生物学上有很多例子 过度磷酸化区域，它们与受体/配体相互作用相关， 包括最常见的 G 蛋白偶联受体 (GPCR)、膜受体 FDA 批准的药物的目标。用于蛋白质的准确位点特异性定量 过度磷酸化我们提出了一种使用超高分辨率离子的跨学科方法 淌度分离 (IMS) 与高精度和灵敏的 MS 和 MS/MS 谱图集成 能够对高度磷酸化的 GPCR 整体进行可靠的表征 提高灵敏度和速度。我们将使用多层结构进行无损离子操作 (SLIM) 技术（SLIM-Orbitrap 平台）可全面表征 利用 CXCR3 的 GPCR/拮抗剂相互作用，CXCR3 在炎症中发挥核心作用 作为初始测试案例，通过调节 T 细胞功能来治疗疾病。我们计划首先整合 超高分辨率 IMS 具有先进的 Orbitrap MS 平台，可明确解码 过度磷酸化位点，评估 SLIM-Orbitrap MS 平台的解析能力 过度磷酸化的蛋白质区域，最后进行全面的位点特异性 通过筛选活化的 T 细胞进行 GPCR 的磷酸蛋白质组学，其剂量反应为 趋化因子和小分子 CXCR3 偏向激动剂。